PROJECT SUMMARY/ABSTRACT The 3-dimensional organization of our genome has emerged as an important regulator of diverse nuclear processes, ranging from gene expression to DNA replication. A wide variety of tools have been useful for the genome-wide study of the 3D genome organization, and these assays generally resolve chromatin topology by detecting the frequency of ligation between proximal genomic fragments in the formaldehyde fixed cells. While these techniques have uncovered general features of chromatin organization in eukaryotic cells, they also produced widely different results that have clouded our view of chromatin organization. In addition, the modest resolution and the biases introduced by restriction digestion and ligation complicate the data interpretation. Here, we propose innovative solutions to address each of these barriers. In aim 1, we will combine genetics, biochemistry and microscopy to develop a gold standard dataset for evaluating and optimizing technologies mapping chromatin topology. Specifically, we will assess long-range chromatin interactions at ~100 pairs of enhancer/promoter loci in an experimental model cell system, with the use of genome editing tools, locus- specific 4C and 3D-FISH, to establish a set of positive and negative controls. In aim 2, we will optimize and refine existing genome wide approaches for assessing high-resolution chromatin structure, guided by the gold standard data generated in aim 1. In aim 3, we will develop and refine a complementary method, termed Genome Architecture Mapping (GAM), that can probe chromatin structure genome-wide without the need for restriction and ligation. This method avoids the potential bias of previous methodologies and also offers the opportunity for analysis of chromatin structure in single nuclei. If successful, the tools and resources developed through this component will transform our ability to study chromatin topology in mammalian cells.